Polymorphism of human platelet membrane glycoprotein IIb and diagnostic applications thereof

ABSTRACT

Isolated polynucleotide molecules and peptides encoded by these molecules can be used in the analysis of alloantigen phenotypes, as well as in diagnostic and therapeutic applications, relating to human platelet Bak polymorphism. By analyzing genomic DNA or amplified genomic DNA, or amplified cDNA derived from platelet mRNA, it is possible to type glycoprotein GPIIb with regard to the Bak polymorphism, for example, in the context of diagnosing and treating clinical syndromes associated with GPIIb-related immune responses.

This invention was made with Government support under grant R29 HL38166-02, "Biochemical Studies of Human Platelet Alloantigens," and grant R37-HL-13629, "Hemorrhagic Diseases," awarded by the National Institutes of Health. The Government has certain rights in the invention.

BACKGROUND OF THE INVENTION

The present invention relates to isolated polynucleotide molecules useful for analyzing alloantigen phenotypes, to peptides encoded by these molecules, and to the diagnostic and therapeutic uses thereof relating to a human platelet Bak polymorphism, including a method for typing platelet membrane glycoproteins which entails an analysis of amplified cDNA produced from platelet mRNA or of genomic DNA.

Blood obtained from different individuals has been found to have different antigenic and immune properties, to the extent that antibodies in the blood of one person may react with antigens on red blood cells or platelets in the blood of another individual. These antigens are often found on membrane glycoproteins present on the surface of the cells. These membrane glycoprotein antigens can induce the production of antibodies against them when they are introduced as foreign proteins in transfused blood or in fetal blood. Human platelets and red blood cells contain dozens of identifiable membrane glycoprotein constituents, only some of which have been well characterized.

Membrane glycoproteins which induce antibody production in the same species are called "alloantigens." Alloantigens have been characterized for both red blood cells and platelets. Recognized classes of red blood cell and platelet alloantigens have been described, over the past 30 years, based on observations of antibody reactions occurring when patients have been exposed to blood from other individuals. The lack of sequenceable antigen protein and clonable antigen-encoding mRNA has prevented molecular characterization of the different alleles coding for many clinically important alloantigens.

One system of alloantigens, consisting of the platelet Bak^(a) and Bak^(b) alloantigens, are carried by the human platelet membrane glycoprotein IIb-IIIa (GPIIb-GPIIIa) complex, which mediates platelet aggregation by providing functional receptors for fibrinogen on platelet surfaces. See Phillips, et al., Blood 71:831-43 (1988). GPIIb and GPIIIa are known to bear a number of clinically important, alloantigenic determinants which are responsible for eliciting an immune response in two well-described clinical syndromes, post-transfusion purpura (PTP) and neonatal alloimmune thrombocytopenia (NATP). See Kunicki & Newman in CURRENT STUDIES IN HEMATOLOGY AND BLOOD TRANSFUSION 18-32 (1986); Aster in ADVANCES IN IMMUNOLOGY AND BONE MARROW TRANSPLANTATION 103-118 (1984).

The Bak alloantigen system is the second or third most frequently implicated stimulus in these disorders. There are two serologically defined, but molecularly undefined, allelic forms of the Bak alloantigen, designated "Bak^(a) " and "Bak^(b)," which are thought to be expression products of the GPIIb gene. von dem Borne, et al., Vox Sang. 39:113 (1980); Kickler, et al., Blood: 71(4):894 (1988); Keifel, et al., Vox Sang. 56:93 (1989). The gene frequencies for these two alleles have been calculated to be 61% for Bak^(a) and 39% for Bak^(b) while the observed phenotypic frequencies are 37% for Bak^(a) homozygous, 15% for Bak^(b) homozygous, and 48% for heterozygous individuals. see Kickler, et al., Vox Sang. 56:93 (1989). Based upon these frequencies, the probability of fetal-maternal Bak incompatibility would be significant, but fewer than 5% of the cases of NATP (or 1/40,000) are attributable to Bak. This suggests that other factors contribute to the likelihood of developing NATP.

Determination of the amino acid sequence variations that are presumably responsible for forming the relevant epitopes of red blood cell and platelet alloantigens has been achieved in only a few instances, due largely to the formidable difficulties in obtaining protein-sequence information from those often large glycoproteins. In particular, the amino acid-sequence variation responsible for the relevant epitopes has not yet been reported for either the Bak^(a) or Bak^(b) forms of the 125 kilodalton (kd) GPIIb molecule.

SUMMARY OF THE INVENTION

It is therefore an object of the present invention to provide polynucleotide molecules that can be used in analyzing Bak alloantigen.

It is also an object of the present invention to provide for the typing of human platelets, based on information obtained through the analysis of nucleic acids, including genomic DNA and cDNA derived from platelets, respectively.

It is yet another object of the present invention to provide ready means for determining platelet Bak alloantigen phenotype.

It is still a further object of the present invention to provide polypeptide molecules for use in generating antibodies that distinguish between the different forms of GPIIb which constitute the Bak polymorphism.

Another object of the present invention is to provide methods for diagnosing and treating clinical syndromes related to a GPIIb-related immune response.

In achieving these objects, there has been provided, in accordance with one aspect of the present invention, an oligonucleotide probe molecule that hybridizes to a portion of the GPIIb gene, which portion includes a nucleotide corresponding to nucleotide 2622 of GPIIb cDNA, where the molecule hybridizes to the portion in question when nucleotide 2622 is guanylate, for one type of allele-specifc probe, or thymidylate for another type. In a preferred embodiment, the oligonucleotide probe of the present invention is is between about ten and thirty bases in length.

In accordance with another aspect of the present invention, a kit for typing platelet Bak alloantigens has been provided that comprises

(a) a receptacle containing a solution of a labeled oligonucleotide probe that distinguishes an allele of a platelet Bak alloantigen from other alleles, or

(b) a receptacle containing a solution of an antibody that discriminately binds a Bak^(a) allele or a Bak^(b) allele of GPIIb, where the antibody (i) recognizes a polypeptide molecule encoded by a nucleotide sequence encoding at least amino acid 843 of GPIIb and (ii) binds either the Bak^(a) allele or the Bak^(b) allele of GPIIb, or

(c) a receptacle containing a solution of an endonuclease recognizing a cleavage site that distinguishes a nucleotide sequence of an allele of a platelet Bak alloantigen from other alleles, and

(d) means for amplifying DNA that comprises at least a portion of a GPIIb gene or GPIIb cDNA, where the portion in question includes a nucleotide corresponding to nucleotide 2622 of GPIIb cDNA.

There has also been provided, in accordance with another aspect of the present invention, a method of typing glycoprotein IIb, comprising the steps of (A) synthesizing cDNA from human platelet mRNA of an individual; (B) amplifying the cDNA to produce amplified cDNA; and then (C) analyzing the amplified cDNA to determine Bak alloantigen phenotype for that individual. In one preferred embodiment, the further comprises synthesizing cDNA from human platelet mRNA of a second individual, repeating aforementioned steps (B) and (C) on the cDNA of second individual, and thereafter discriminating between the first and second individuals based on the alloantigen phenotype. In another preferred embodiment, step (C) comprises the steps of (i) digesting the amplified cDNA with a restriction endonuclease recognizing a cleavage site that distinguishes a nucleotide sequence of a first Bak allele from another Bak allele; and then (ii) analyzing the cDNA fragments to determine the Bak alloantigen phenotype.

In accordance with yet another aspect of the present invention, a method of typing platelet Bak membrane glycoproteins has been provided that comprises the steps of (A) obtaining genomic DNA from an individual and (B) analyzing the genomic DNA to determine a platelet Bak alloantigen phenotype. In a preferred embodiment, step (B) comprises (i) digesting the genomic DNA with a restriction endonuclease to produce DNA fragments; thereafter (ii) hybridizing the DNA fragments with a labeled, allele-specific oligonucleotide probe that distinguishes a nucleotide sequence of an allele of a platelet Bak alloantigen from other alleles; and then (iii) analyzing the probe that has hybridized to the DNA fragments in order to determine the Bak alloantigen phenotype.

In accordance with still another aspect of the present invention, there has been provided a method of typing platelets with respect to GPIIb that comprises the steps of (A) obtaining genomic DNA from an individual, (B) amplifying the genomic DNA to produce amplified genomic DNA and (C) analyzing the amplified genomic DNA to determine a platelet Bak alloantigen phenotype. In a preferred embodiment, step (C) comprises of (i) hybridizing the amplified genomic DNA with a labeled, allele-specific oligonucleotide probe that distinguishes a nucleotide sequence of a first Bak allele from that of another Bak allele; and then (ii) analyzing the probe that has hybridized to the amplified genomic DNA to determine said alloantigen phenotype. In another preferred embodiment, step (C) comprises

(i) hybridizing the amplified genomic DNA with a pair of oligonucleotide probes to form a construct, wherein a first probe of the pair of probes is labeled with a first label and the other probe is labeled with a second label, such that the first label is distinguishable from the second label, and the probes hybridize adjacently to each other at a nucleotide that distinguishes a Bak allele from another Bak allele; thereafter

(ii) reacting said construct with a ligase in a reaction medium; and then

(iii) analyzing said reaction medium to detect the presence of a ligation product comprising the first probe the said second probe.

A polypeptide molecule is further provided, in accordance with another aspect of the present invention, that comprises an amino-acid sequence that corresponds to a tetramer fragment of GPIIb, wherein the fragment comprises amino acid 843 of GPIIb and wherein the molecule is not GPIIb itself. Preferably, the polypeptide molecule is between four and fifty amino-acid residues in length. In addition, it is preferred that the polypeptide molecule is itself immunogenic or is attached to a immunogenicity-imparting carrier, forming another molecule of the present invention.

According to another aspect of the present invention, an antibody is provided that distinguishes the Bak^(a) form of GPIIB antigen from the Bak^(b) form, where the antibody recognizes a polypeptide sequence that comprises at least amino acid 843 of GPIIb. The antibody can be a monoclonal antibody produced by a method comprising the steps of (A) immunizing a mammal with an antigenic molecule comprising a polypeptide as described above, then (B) removing lymphocytes from the mammal, (C) fusing the lymphocytes with mammalian myeloma cells to form hybridoma cells, (D) culturing the hybridoma cells and thereafter (E) selecting, isolating and cloning hybridoma cells secreting monoclonal antibodies that distinguish between the Bak^(a) and Bak^(b) forms of GPIIb.

A method is also provided, pursuant to another aspect of the present invention, for treating post-transfusion purpura or neonatal alloimmune thrombocytopenia, comprising the step of administering to an individual a formulation comprised of a peptide in a pharmacologically effective concentration and a physiologically-compatible carrier therefor, where the individual (i) suffers from post-transfusion purpura or is the mother of a fetus at risk for developing NATP and (ii) has anti-Bak^(a) or anti-Bak^(b) antibodies, said peptide binding an antibody selected from the group consisting of an anti-Bak^(a) antibody and an anti-Bak^(b) antibody.

In accordance with yet another aspect of the present invention, an isolated DNA molecule has been provided that comprises a nucleotide sequence corresponding to a portion of the GPIIb gene that includes a nucleotide corresponding to nucleotide 2622 of GPIIb cDNA, wherein the molecule is not coincident with the GPIIb gene.

Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagrammatic representation of the GPIIb mRNA molecule. The locations of two oligonucleotide primers used for PcR amplification are also shown.

FIG. 2 shows autoradiographs of electrophoretic gels used in the sequence analysis of amplified GPIIb cDNA, derived from both a Bak^(a) homozygous individual and a Bak^(b) homozygous individual. A segment of the autoradiograph, encompassing bases 2615 to 2626 indicates a single base substitution of a thymine (T) (Bak^(b) allele) for a guanine (G) (Bak^(a) allele) at base 2622. (See below regarding the number of nucleotides herein.)

FIG. 3 shows an autoradiograph of the results of an analysis of Bak phenotype by allele-specific oligonucleotide hybridization. Bases 1988 to 2821 were enzymatically amplified from platelet RNA from nine individuals of known Bak phenotype. An allele-specific oligonucleotide (Probe A) hybridized to DNA from Bak^(a) homozygous individuals (wells 1-4). A second allele-specific oligonucleotide (Probe B) hybridized to DNA from Bak^(b) homozygous individuals (wells 5-7). Heterozygous individuals were positive with both probes (wells 8 and 9).

FIG. 4 is the amino acid sequence of the region of GPIIb responsible for the Bak polymorphism.

FIGS. 5A-5M present the prior art cDNA and corresponding amino acid sequences for the platelet membrane glycoprotein IIb as reported and numbered in Poncz et al., J. Biol. Chem. 262:8476 (1987).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

It has been discovered that a single nucleotide of GPIIb is responsible for the Bak polymorphism. In light of this discovery, manipulation of nucleic-acid molecules derived from platelets can be effected to provide for the analysis of alloantigen phenotypes, for the generation of peptides encoded by these molecules, and for the use of such peptides in diagnosis and therapy relating to a human platelet Bak polymorphism. Nucleic-acid molecules utilized in these contexts may be amplified, as described below, and generally include RNA, genomic DNA and cDNA derived from RNA.

Although the generation of cDNA from platelet or red blood cell mRNA was previously thought to be unfeasible, a new approach has been discovered for examining platelet mRNA from single individuals. As described in copending U.S. application Ser. No. 07/343,827, the contents of which are hereby incorporated by reference, is been found that mRNA can be obtained from platelets as well as red blood cells in quantities sufficient for isolation, cDNA generation, and amplification. By generating and amplifying cDNA produced from mRNA of a number of individuals of known platelet allotypes, the nucleotide sequence variations that exist in the genes that express alloantigen determinants can be ascertained. Furthermore, by isolating and amplifying mRNA from a number of individuals of known allotype, it is possible, pursuant to the present invention, to identify phenotype-specific nucleotide sequence variations in corresponding genes.

To obtain amplified cDNA from platelet mRNA, mRNA derived via conventional methods, see, e.g., MANIATIS, ET AL., MOLECULAR CLONING: A LABORATORY MANUAL 187-210 (Cold Spring Harbour Laboratory, 1982), from platelets can be converted to cDNA and then enzymatically amplified to produce microgram quantities of platelet-specific cDNA. This amplification is preferably accomplished via the "polymerase chain reaction" (PcR) method disclosed by U.S. Pat. Nos. 4,683,195 and 4,800,159, the respective contents of which are hereby incorporated by reference.

More specifically, in the process of generating and amplifying cDNA encoded by the isolated platelet mRNA, oligonucleotide primer pairs can be constructed that allow enzymatic amplification of a cDNA segment obtained from an mRNA molecule that encodes an amino-acid sequence defining the polymorphism. The corresponding, isolated cDNAs can then be analyzed to determine the molecular basis of observed phenotypic differences. The ability to compare directly the nucleotide and corresponding amino-acid sequences of genes encoding alleles of alloantigens is made possible by (1) the discovery that cDNA can be generated and amplified successfully from platelet mRNAs and (2) the determination of a nucleotide sequence of a glycoprotein which is thought to be polymorphic.

The molecular description of polymorphisms associated with platelet alloantigens can be provided by analyzing amplified cDNA, generated from platelet mRNA, according to one of the following methods: differential restriction endonuclease digestion (DRED), allele-specific oligonucleotide probing (ASOP), and ligase-mediated gene detection (LMGD). Additional methods of analysis would also be useful in this context, such as fluorescence resonance energy transfer (FRET) as disclosed by Wolf, et al., Proc. Nat. Acad. Sci. USA 85: 8790-94 (1988), the contents of which are hereby incorporated by reference.

DRED analysis is accomplished in the following manner. If conditions occur including (1) a particular amplified cDNA segment contains a sequence variation that distinguishes an allele of a polymorphism and (2) this sequence variation is recognized by a restriction endonuclease, then the cleavage by the enzyme of a particular polynucleotide segment can be used to determine the alloantigen phenotype. In accomplishing this determination, amplified cDNA derived from platelet mRNA is digested and the resulting fragments are analyzed by size. The presence or absence of nucleotide fragments, corresponding to the endonuclease-cleaved fragments, determines which phenotype is present.

Thus, a guanine(G) thymine(T) polymorphism at base 2622 is revealed by examination of the nucleotide sequence contained in cDNA generated from mRNA derived from Bak^(b) -homozygous vs. Bak^(a) -homozygous individuals. (Throughout this description, the numbering of nucleotides in mRNAs and cDNAs is with reference to the cDNA sequence disclosed by Poncz, et al., J. Biol. Chem. 262:8476 (1987), the contents of which article are hereby incorporated by reference. See FIGS. 5A-5M. A nucleotide of genomic DNA that corresponds to a particular nucleotide in a cDNA is designated by the number of the cDNA nucleotide.) This single nucleotide substitution results in the creation of a unique restriction enzyme cleavage site for the restriction endonuclease FokI. By utilizing a restriction endonuclease with the selectivity of FokI or an isoschizimer thereof to discriminate between these two polymorphic sequences, the phenotypes of individuals can be determined in the above-described manner. Sequence analysis of the resulting restriction fragments demonstrates that the Bak^(b) form of GPIIb mRNA contains the codon AGC, encoding serine at position 843 of the known GPIIb amino-acid sequence, in place of an ATC codon coding for isoleucine at position 843 in the Bak^(a) form. (The designation of amino acid residues in this regard follows the numbering system of Poncz, et al., incorporated above by reference. See FIGS. 5A-5M.)

In ASOP analysis according to conventional methods, oligonucleotide probes are synthesized that will hybridize, under appropriate annealing conditions, exclusively to a particular amplified cDNA segment that contains a nucleotide sequence that distinguishes one allele from other alleles of a platelet membrane glycoprotein. Such a probe would be discernably labeled so that when it hybridizes to the allele-distinguishing cDNA segment, it can be detected and the specific allele thus identified.

For example, an oligonucleotide probe can be synthesized, in accordance with the present invention, that will hybridize to a cDNA segment, derived from GPIIb mRNA, that contains the base thymine at polymorphic nucleotide 2622 (nucleotide=thymidylate). Alternatively, an oligonucleotide probe of the present invention will hybridize what the cDNA segment contains the base guanine at nucleotide 2622 (nucleotide=guanylate). These allele-specific probes can be appropriately labeled and added to the generated cDNA segments under annealing conditions, such that one of the allele-specific probes hybridizes and can be detected, thereby identifying the specific BAk^(a) or Bak^(b) allele. In accordance with conventional procedure, the design of an oligonucleotide probe according to the present invention preferably involves adjusting probe length to accommodate hybridization conditions (temperature, ionic strength, exposure time) while assuring allele-specificity. A length of ten to thirty nucleotides is typical.

In the course of the third method of analysis, LMGD, as disclosed by Landegren, et al., Science 241:1077-80 (1988), the contents of which are hereby incorporated by reference, a pair of oligonucleotide probes are synthesized that will hybridize adjacently to each other, i.e., to a cDNA segment under appropriate annealing conditions, at the specific nucleotide that distinguishes one allele from other alleles of a platelet membrane glycoprotein. Each of the pair of specific probes is labeled in a different manner, and, when both probes hybridize to the allele-distinguishing cDNA segment, the probes can be ligated together by the addition of a ligase.

When the ligated probes are separated and isolated from the cDNA segments, both types of labeling can be observed together on a Northern blot when analyzed by conventional procedures, confirming the presence of the allele-specific nucleotide sequence. Where the above-described pair of differently labeled probes bind to a nucleotide sequence containing a distinguishing nucleotide of a different allele, the probe pair is not ligatable and, after the probes are isolated from the cDNA segments, each type of labeling is observed to be separate from the other label type.

An exemplary LMGD analysis, according to the present invention, entails the use of a pair of oligonucleotide probes, wherein one probe is radioactively ³² p-labeled and the other probe is biotin-labeled. Under appropriate conditions, the pair of probes adjacently hybridizes to a cDNA segment at a nucleotide corresponding to nucleotide 2622 of GPIIb. The biotin labeled probe hybridizes to nucleotides 2602-2622 of GPIIb, wherein nucleotide 2622 contains a thymine, which distinguishes the Bak^(b) allele. The ³² p-labeled probe hybridizes nucleotides 2623-2633 of GPIIb and, therefore will hybridize adjacently to the biotin-labeled probe. These probes are then added under annealing conditions such that they hybridize adjacently to each other spanning nucleotides 2602-2633 of GPIIb. The biotin labeled probe is detected by the binding of the compound strepavidin after hybridization and the p³² -labeled probe is detected by autoradiography, according to conventional procedures.

When the Bak^(b) allele sequence is present in the amplified cDNA, then the addition of a ligase will result in the biotin labeled probe being covalently bound to the ³² p-labeled probe. The ligation is possible, because the ends of the probes that are adjacent to each other (hybridized to nucleotides (2622 and 2633) are both hybridized to the cDNA. In the case where these two probes hybridize to the Bak^(b) allelic form of the cDNA sequence, the biotin-labeled probe end at nucleotide 2622 is not hybridized appropriately, preventing the ligation step from occurring. When this pair of probes binds completely to the Bak^(b) allele sequence, therefore, the probes are ligated and when the probes are separated from the Bak^(b) sequence and exposed so as to be detected, both the biotin/strepavidin and the ³² p labeling are present together. When the Bak^(a) allele sequence is hybridized, on the other hand, the probes cannot be ligated, and the biotin/strepavidin- and ³² p-labeling are observed separately. In this manner, the Bak^(b) and Bak^(a) alleles sequences and corresponding phenotype can be distinguished.

Alternatively, DRED, ASOP and LMGD or other suitable methods of analysis, such as FRET, can be used with genomic or amplified-genomic DNA to distinguish platelet membrane glycoprotein Bak^(b) from Bak^(a), starting with any nucleated cell sample, obtained from an individual, from which DNA can be isolated in sufficient quantities for analysis. Amplified genomic DNA would be amplified from isolated genomic DNA in the same manner as described above for cDNA. Once a tissue sample, such as cells scraped from the inside of an individual's cheek, has been obtained, genomic DNA isolated by conventional procedures can be analyzed directly per se or amplified prior to analysis.

The foregoing description of the three types of analysis would apply to the use of genomic DNA or amplified-genomic DNA, with the term "cDNA" being replaced with "genomic or amplified genomic DNA." One difference in the analysis of genomic DNA or amplified genomic DNA is that the GPIIb sequence used for designing a suitable oligonucleotide probe might have to include any intronic sequences, which would not be present in the cDNA of GPIIb, that were near or adjacent to the nucleotide that determines the Bak phenotype.

In general, the presence of intronic sequences near the phenotype-determining nucleotide can be ascertained by sequence analysis of genomic DNA, accomplished via Maxam-Gilbert or another conventional technique. Sequence information on the region of genomic DNA encompassing an exon that encodes the polymorphism can be used to design appropriate oligonucleotides, such that a genomic DNA-based PcR could be performed. The resulting amplified products can then be assessed for alloantigen phenotype, in accordance with the present invention, by means of any of the above-described diagnostic methods.

The polymorphic nucleotide which distinguishes the above-described GPIIb alleles is located (see asterisk) in an exon shown below with flanking genomic segments. In accordance with convention, the following is the coding sequence of the genomic DNA; the GPIIb amino-acid residues encoded by the exon are also shown, with conventional acronyms used (v for valine, d for aspartic acid, etc.): ##STR1## (Sequence data provided by Dr. Mortimer Poncz, The Children's Hospital of Philadelphia.) More generally, the primers used for PcR amplification should be positioned, relative to the exon which contains the polymorphic nucleotide, so that the amplified region encompasses that nucleotide, which corresponds to base 2622 of the GPIIb cDNA. For example, the solid bar and striped bar above denote, respectively, a sequence of a first primer and the complementary sequence of a second primer which are suitable for genomic amplification as described herein.

The ability to perform DNA-typing analysis for determination of Bak phenotypes, pursuant to the present invention, has a number of useful clinical applications, including but not limited to those involving determination of the Bak alloantigen phenotype of an individual, and the diagnosis and treatment of a pathological immune response (or potential response) involving foreign alloantigens or antibodies. In accordance with the present invention, alloantigen phenotyping can be effected by the generation of amplified genomic DNA or amplified cDNA from platelet mRNA, permitting diagnosis of individuals for the purpose of treating or preventing pathological immune responses.

Once the nucleotide-sequence variations specific for each allelic form of the alloantigens of a given class are determined, other conventional methods can be employed, through the use of genomic DNA or platelet RNA, to perform the same type of diagnosis on other individuals. These methods would include, but not are limited to, allele-specific nucleotide probing and ligase-mediated gene detection, as previously described.

Diagnostic kits can also be used, in accordance with the present invention, for the determination and diagnosis of alloantigen phenotypes via the procedures described herein. Such a kit can include, inter alia, antibodies or antibody fragments to an antigenic determinant expressed by either of the above-described Bak^(a) - and Bak^(b) -encoding sequences, which antibodies would react with the blood sample of an individual so as to indicate whether that individual has a Bak^(a) or Bak^(b) phenotype. Alternatively, all the reagents required for the detection of nucleotide(s) that distinguish the Bak alloantigens, by means described herein, can be provided in a single kit that uses isolated genomic DNA or platelet mRNA from an individual. Containing a labeled probe that distinguishes, for example, nucleotide 2622 of GPIIb, such a kit can be utilized for Bak alloantigen phenotyping.

A further beneficial use of the nucleotide sequences that distinguish the Bak^(a) allele from the Bak^(b) allele is to obtain or synthesize the respective expression product, in the form of a polypeptide, encoded by these nucleotide sequences. These polypeptides can be used to generate antibodies for diagnostic and therapeutic uses, for example, with regard to pathological conditions such as PTP or NATP.

A polypeptide within the present invention which can be used for the purpose of generating such antibodies preferably comprises an amino-acid sequence that corresponds to (i.e., is coincident with or functionally equivalent to) a four-residue (tetramer) fragment of the GPIIb molecule that includes amino acid 843. When the latter amino acid is serine, the polypeptide can be used, as described above, to produce antibodies that specifically bind the Bak^(b) form of GPIIb; when it is isoleucine, antibodies can be obtained that particularly recognize the Bak^(a) form. The class of polypeptides thus defined, in accordance with the present invention, is not intended to include the GPIIb molecule itself, but does encompass fragments of the molecule as well as synthetic polypeptides meeting the aforementioned definition.

Although the length of a polypeptide within this class is not critical, the requirement for immunogenicity may require that the polypeptide be attached to a immunogenicity-imparting carrier, e.g., a particulate carrier like a liposome or a soluble macromolecule (protein or polysaccharide) with a molecular weight in the range of about 10,000 to 1,000,000, or be administered with an adjuvant, such as complete Freund's adjuvant. For artificial polypeptides, as distinguished from GPIIb fragments, maximum length is determined largely by the limits of techniques available for peptide synthesis, say, about fifty amino acids. Thus, a synthetic polypeptide of the present invention is preferably between four and about fifty amino acids in length.

In this context, the term "antibody" encompasses monoclonal and polyclonal antibodies. Such an antibody can belong to any antibody class (IgG, IgM, IgA, etc.). For monoclonal antibody (Mab) production, one generally proceeds by isolating lymphocytes and fusing them with myeloma cells, producing hybridomas. The cloned hybridomas are then screened for production of antibodies the bind preferentially to either the Bak^(a) form or the Bak^(b) form of GPIIb. "Antibody" also encompasses fragments, like Fab and F(ab')₂, of anti-Bak^(a) or anti-Bak^(b) antibodies, and conjugates of such fragments, and so-called "antigen binding proteins" (single-chain antibodies) which are based on anti-Bak^(a) or anti-Bak^(b) antibodies, in accordance, for example, with U.S. Pat. No. 4,704,692, the contents of which are hereby incorporated by reference. Human alloantisera currently used for serological typing are specifically excluded from this definition. Alternatively, Mabs or a fragment thereof within the present invention can be produced using conventional procedures via the expression of isolated DNA which codes for variable regions of such an Mab in host cells like E. coli, see, e.g., Ward, et al., Nature, 341:544-546 (1989), or transfected murine myeloma cells. See Gillies, et al., Biotechnol. 7: 799-804 (1989); Nakatani, et al., Biotechnol. 7:805-10 (1989).

Diagnostic applications of these antibodies are exemplified, according to the present invention, by the use of a kit containing an anti-Bak^(a) or an anti-Bak^(b) antibody which undergoes a reaction with a sample of an individual's blood to determine a Bak^(a) or Bak^(b) platelet phenotype. Such a reaction involves the binding of anti-Bak^(a) antibody to Bak^(a) antigen or the binding of anti-Bak^(b) antibody to Bak^(b) antigen. The observation antibody-antigen complex in a blood sample would indicate a positive result. A kit of this sort could be used to diagnose, or to help prevent, the occurrence of pathological conditions like PTP or NATP.

A polypeptide of the present invention that is recognized specifically by anti-Bak^(a) or anti-Bak^(b) antibodies can be used therapeutically. Thus, antibodies raised against such a polypeptide can employed in the generation, via conventional methods, of anti-idiotypic antibodies (that is, antibodies that bind an anti-Bak^(a) or anti-Bak^(b) antibody), e.g., by the use of hybridomas as described above. See, for example, U.S. Pat. No. 4,699,880, the contents of which are hereby incorporated by reference. Such anti-idiotypic antibodies would bind endogenous or foreign anti-Bak antibodies in the blood of an individual, thereby to treat or prevent pathological conditions associated with an immune response to a "foreign" Bak alloantigen. Alternatively, a polypeptide within the present invention can be administered, with a physiologically-compatible carrier, to achieve the same qualitative effect, namely, the selective reduction or elimination of circulating anti-Bak antibodies from a patient suffering or at risk from an immune response.

The present invention is further described below by reference to the following, illustrative examples. Used in the examples were platelet samples from four homozygous Bak^(a) individuals, three homozygous Bak^(b) individuals, and two individuals who were heterozygous for the Bak allotype. The respective phenotypes of all the test subjects had been identified using well-characterized anti-Bak^(a) and anti-Bak^(b) human alloantisera.

EXAMPLE 1: AMPLIFICATION OF cDNA

Platelet RNA from a panel of nine normal volunteers, including four Bak^(a/a), three Bak^(b/b) and two Bak a/b individuals, was prepared according to the procedure developed by Chomczynski and Sacchi, Anal. Biochem. 162:156 (1987), except that the final RNA pellet was subjected to one additional phenol/chloroform extraction and ethanol precipitation necessary to achieve reproducible gene amplification of platelet cDNA. Bak^(a) and Bak^(b) phenotype was assessed using well-characterized human alloantisera in a standard antigen capture assay, see Furihata, et al., J. Clin. Invest. 80:1624 (1987); Chomczynski and Sacchi, Anal. Biochem. 162:156 (1987). The C-terminal end of the GPIIb heavy and light chain message from base 1988 to 2821 was selected for sequence analysis and comparison, and two 24-base oligonucleotide primers flanking 833 base pairs of this region were synthesized on a Gene Assembler (Pharmacia Fine Chemicals, Piscataway, N.J.).

The anti-sense primer (5'-CAGGAAGGCCAGCACCGTGACCATG-3') from base 2821 to 2797 was used to prime the synthesis of cDNA from platelet RNA as previously described (Newman, et al., J. Clin. Invest. 82:739 (1988); Newman, et al., J. Clin. Invest. 83:1778 (1989). The second strand was generated by the sense primer (5'GAGCTGCAGATGGACGCAGCCAAC-3') from base 1988 to 2011 during the first round of PcR. Amplification was carried out in a DNA Thermal Cycler (Perkin-Elmer Cetus, Norwalk, Conn.) programmed to permit denaturation at 94° C. for on 1/2-minute, annealing at 50° C. for one 1/2-minute, and chain extension at 72° C. for three minutes. The reaction was allowed to proceed for 30 cycles followed by a final incubation at 72° C. for seven minutes to allow completion of strand synthesis.

EXAMPLE 2: ANALYSIS OF PcR PRODUCTS

PcR samples were analyzed on 1.8% Seakem GTG agarose gels (FMC BioProducts, Rockland, Me.), and the appropriate bands were excised and recovered by electroelution. The plasmid vector pGEM-5Zf (Promega Biotech, Madison, Wis.) was prepared for ligation by restriction digestion with Eco RV (New England Biolabs, Beverly, Mass.) to yield blunt ends, and ligated to purified amplification product, followed by transformation into E. coli strain NM522 competent cells (Stratagene Cloning Systems, San Diego, Calif.). Two clones representing each Bak homozygous phenotype were selected for direct sequence analysis of the plasmid DNA by the dideoxy sequencing method using T7 DNA polymerase (USB, Cleveland, Ohio USA). Four 24 or 25 base oligonucleotides were synthesized and used as sequencing primers.

The results (shown in FIG. 2) demonstrated that a single nucleotide difference was observed between the Bak^(a/a) and Bak^(b/b) clones at base 2622. Analysis of the cDNA derived from the Bak^(a/a) individual revealed that thymine was present at this position, whereas guanine was substituted in this position in the Bak^(b/b) cDNA. This resulted in a substitution of a serine for an isoleucine at amino acid residue 843.

EXAMPLE 3: ALLELE-SPECIFIC HYBRIDIZATION

Amplified cDNA from four individuals with Bak^(a/a) phenotype, three with Bak^(b/b), and two heterozygous for Bak was subjected to hybridization with 13-base allele-specific oligonucleotides (ASO). Probe A (TGCCCATCCCCAG) corresponds to the published sequence of GPIIb (Poncz, et al., J. Biol. Chem. 262(18):8476 (1987)) from base 2616 to 2628, while Probe B (TGCCCAGCCCCAG) differs only in the middle base, a G instead of a T, and corresponds to a single base difference observed in the region sequenced. The probes (200 ng) were end-labeled with digoxigenin-11-dUTP (Boehringer Mannheim, Indianapolis, Ind.) in 25 μl 100 mmol/L potassium cacodylate, 2 mmol/L CoCl₂, 0.2 mmol/L DTT, pH 7.2 containing 1 U terminal transferase (Boehringer Mannheim, Indianapolis, Ind.), and the probes were used for hybridization without purification. Amplified DNA was used directly for blotting or, in some cases, appropriate bands were recovered from agarose gels using Gene Clean (Bio 101, LaJolla, Calif.). The samples were eluted in 20 μl water, diluted 1/10,000, and 10 μl was used for reamplification using the same probes and PcR conditions. Amplified or reamplified DNA was denatured in 0.25 N NaOH, 1.5 mol/L NaCl at room temperature for 15 minutes. Each sample was divided between two wells of a Minifold dot blot apparatus (Schleicher and Schuell, Keene, N.H.) and transferred to Magnagraph nylon membrane (MSI, Westboro, Mass.) by vacuum suction. The filter was exposed to UV irradiation (Fotodyne, New Berlin, Wis.) for 5 minutes followed by baking at 80° C. for 15 minutes. The membrane was prehybridized in 5X Denhardt's, 5X SSC, 10 mmol/L EDTA, 10 mmol/L Na₂ HPO₄, pH 7 at 68° C. for one hour, and then cut into two strips which were hybridized to either Probe A or Probe B in 4 mls 10X Denhardt's 5X SSC, 5 mmol/L EDTA, 7% SDS, 50 ug/ml Salmon sperm DNA, 20 mmol/l Na₂ HPO₄, pH 7 at 42° C. overnight. The filters were washed in 2 changes 6X SSC for 30 minutes each at room temperature followed by 2 changes of 3 mol/L tetramethylammonium chloride (Aldrich Chemical, Milwaukee, Wis.), 2 mmol/L EDTA, 1% SDS, 50 mmol/L Tris, pH 8 for 20 minutes each at 42° C. Positive hybridizations using The Genius kit (Boehringer Mannheim, Indianapolis, Ind.) which employs an alkaline phosphatase-conjugated antidigoxigenin antibody, according to the manufacturer's directions.

The results, shown in FIG. 3, demonstrated that Probe A was positive with the four Bak^(a/a) homozygous individuals, Probe B was positive with the three Bak^(b/b) homozygous individuals, and both probes were positive with amplified DNA from the two heterozygous individuals analyzed. 

What is claimed is:
 1. A kit for typing platelet Bak alloantigens, comprising (a) means for amplifying at least a portion of a GPIIb gene, wherein the amplified portion includes the nucleotide corresponding to nucleotide 2622 of the corresponding GPIIb cDNA, and wherein said portion is of sufficient length for identification of said nucleotide corresponding to nucleotide 2622 of the corresponding GPIIb cDNA by hybridization with a labeled oligonucleotide probe or by restriction endonuclease digestion; and (b) at least one component selected from the group consisting of (i) a receptacle containing a solution of a labeled oligonucleotide probe that distinguishes a Bak^(a) -allele nucleotide sequence from a Bak^(b) -allele nucleotide sequence, said sequences comprising a nucleotide of genomic DNA corresponding to nucleotide 2622 of a GPIIb cDNA and (ii) a receptacle containing a solution of a restriction endonuclease which recognizes a cleavage site that distinguishes a Bak^(a) -allele nucleotide sequence from a Bak^(b) -allele nucleotide sequence, said sequences comprising a nucleotide of genomic DNA corresponding to nucleotide 2622 of a GPIIb cDNA.
 2. A kit for typing platelet Bak alloantigens, comprising (a) means for amplifying at least a portion of a GPIIb cDNA, wherein the amplified portion includes nucleotide 2622 of the GPIIb cDNA and wherein said portion is of sufficient length for identification of nucleotide 2622 of the cDNA by hybridization with a labeled oligonucleotide probe or by restriction endonuclease digestion; and (b) at least one component selected from the group consisting of (i) a receptacle containing a solution of a labeled oligonucleotide probe that distinguishes a Bak^(a) -allele nucleotide sequence from a Bak^(b) -allele nucleotide sequence, said sequences comprising the nucleotide at position 2622 of a GPIIb cDNA; and (ii) a receptacle containing a solution of a restriction endonuclease which recognizes a cleavage site that distinguishes a Bak^(a) -allele nucleotide sequence from a Bak^(b) -allele nucleotide sequence, said sequences comprising the nucleotide at position 2622 of a GPIIb cDNA.
 3. A method of typing glycoprotein GPIIb comprising the steps of (A) synthesizing cDNA from mRNA from human platelets of a first individual; (B) amplifying a portion of the cDNA synthesized in step (A) that encodes at least part of glycoprotein GPIIb, said amplified portion containing nucleotide 2622 of the GPIIb cDNA; and (C) analyzing amplified cDNA from step (B) by a method which distinguishes a Bak^(a) -allele nucleotide sequence from a Bak^(b) -allele nucleotide sequence, said sequences comprising the nucleotide at position 2622 of a GPIIb cDNA, in order to determine the Bak allele or alleles carried by said first individual.
 4. A method as claimed in claim 3 further comprising the step of synthesizing cDNA from human platelet mRNA of a second individual, repeating steps (B) and (C) on the cDNA of said second individual, and thereafter discriminating between said first and second individuals based on the Bak alleles carried by them, respectively.
 5. A method as claimed in claim 3 wherein step (C) comprises the steps of (i) preparing cDNA fragments by digesting said amplified cDNA with a restriction endonuclease which recognizes a cleavage site that distinguishes a Bak^(a) -allele nucleotide sequence from a Bak^(b) -allele nucleotide sequence, said sequences comprising the nucleotide at position 2622 of a GPIIb cDNA, and (ii) analyzing said cDNA fragments to determine said Bak allele or alleles.
 6. A method as claimed in claim 3 wherein step (C) comprises the steps of (i) hybridizing said amplified cDNA with a labeled allele-specific oligonucleotide probe that distinguishes a Bak^(a) -allele nucleotide sequence from a Bak^(b) -allele nucleotide sequence, said sequences comprising the nucleotide at position 2622 of a GPIIb cDNA, and then (ii) analyzing the Bak-allele specificity of said probe that has hybridized to said amplified cDNA to determine said Bak allele or alleles.
 7. A method as claimed in claim 3, wherein step (C) comprises the steps of (i) hybridizing said amplified cDNA with a pair of oligonucleotide probes to produce a construct, wherein the first probe of said pair is labeled with a first label and the second probe of said pair is labeled with a second label, such that the first label is distinguishable from the second label, and the probes hybridize adjacently to each other at the nucleotide of the amplified cDNA that corresponds to a nucleotide at position 2622 of a GPIIb cDNA; and thereafter (ii) reacting said construct with a ligase in a reaction medium; and then (iii) analyzing said reaction medium to detect the presence of a ligation product comprising said first probe ligated to said second probe.
 8. A method of typing platelet Bak membrane glycoprotein GPIIb comprising the steps of (A) obtaining from a first human individual genomic DNA which comprises a nucleotide corresponding to nucleotide 2622 of a GPIIb cDNA and (B) analyzing said genomic DNA by a method which distinguishes a Bak^(a) -allele nucleotide sequence from a Bak^(b) -allele nucleotide sequence, said sequences comprising a nucleotide of genomic DNA corresponding to the nucleotide at position 2622 of a GPIIb cDNA, in order to determine the Bak allele or alleles carried by said first individual.
 9. A method as claimed in claim 8 further comprising the step of obtaining from a second human individual genomic DNA which comprises a nucleotide corresponding to nucleotide 2622 of a GPIIb cDNA, repeating step (B) on the genomic DNA of said second individual, and thereafter discriminating between said first and second individuals based on the Bak alleles carried by them, respectively.
 10. A method as claimed in claim 8 wherein step (B) comprises the steps of (i) preparing genomic DNA fragments by digesting said genomic DNA, which comprises a nucleotide corresponding to nucleotide 2622 of a GPIIb cDNA, with a restriction endonuclease which recognizes a cleavage site that distinguishes a Bak^(a) -allele nucleotide sequence from a Bak^(b) -allele nucleotide sequence, said sequences comprising a nucleotide which corresponds to the nucleotide at position 2622 of a GPIIb cDNA, and (ii) analyzing said genomic DNA fragments with a labeled probe to determine said Bak allele or alleles.
 11. A method as claimed in claim 8 wherein step (B) comprises the steps of (i) preparing genomic DNA fragments by digesting said genomic DNA with a restriction endonuclease, provided that said DNA fragments which comprise the nucleotide corresponding to nucleotide 2622 of a GPIIb cDNA are of sufficient length for identification of said nucleotide corresponding to nucleotide 2622 of a GPIIb cDNA by hybridization with a labeled oligonucleotide probe, (ii) hybridizing said DNA fragments with a labeled allele-specific oligonucleotide probe that distinguishes a Bak^(a) -allele nucleotide sequence from a Bak^(b) allele nucleotide sequence, said sequences comprising a nucleotide corresponding to the nucleotide at position 2622 of a GPIIb cDNA, and (iii) analyzing the Bak-allele specificity of said probe that has hybridized to said DNA fragments to determine said Bak allele or alleles.
 12. A method as claimed in claim 8, wherein step (B) comprises the steps of (i) hybridizing said genomic DNA with a pair of oligonucleotide probes to produce a construct, wherein the first probe of said pair is labeled with a first label and the second probe of said pair is labeled with a second label, such that the first label is distinguishable from the second label, and the probes hybridize adjacently to each other at the nucleotide of the genomic DNA that corresponds to a nucleotide at position 2622 of a GPIIb cDNA; and thereafter (ii) reacting said construct with a ligase in a reaction medium; and then (iii) analyzing said reaction medium to detect the presence of a ligation product comprising said first probe ligated to said second probe.
 13. A method of typing platelets to identify a Bak alloantigen phenotype, comprising the steps of (A) obtaining genomic DNA from a first human individual; (B) amplifying a portion of the genomic DNA obtained in step (A) that is at least part of the genomic DNA for glycoprotein GPIIb, said amplified portion containing the nucleotide of genomic DNA corresponding to nucleotide 2622 of the GPIIb cDNA; and (C) analyzing amplified DNA from step (B) by a method which distinguishes a Bak^(a) -allele nucleotide sequence from a Bak^(b) -allele nucleotide sequence, said sequences comprising a nucleotide corresponding to the nucleotide at position 2622 of a GPIIb cDNA, in order to determine the Bak allele or alleles carried by said first individual.
 14. A method as claimed in claim 13 further comprising the step of obtaining genomic DNA from a second human individual, repeating steps (B) and (C) on the genomic DNA of said second individual, and thereafter discriminating between said first and second individuals based on the Bak alleles carried by them, respectively.
 15. A method as claimed in claim 13 wherein step (C) comprises the steps of (i) digesting said amplified portion of genomic DNA with a restriction endonuclease to produce DNA fragments, said restriction endonuclease recognizing a cleavage site in said amplified portion that distinguishes a Bak^(a) -allele nucleotide sequence from a Bak^(b) -allele nucleotide sequence, said sequences comprising a nucleotide corresponding to the nucleotide at position 2622 of a GPIIb cDNA, and (ii) analyzing said DNA fragments to determine said Bak allele or alleles.
 16. A method as claimed in claim 13 wherein step (C) comprises (i) hybridizing said amplified portion of genomic DNA with a labeled allele-specific oligonucleotide probe that distinguishes a Bak^(a) -allele nucleotide sequence from a Bak^(b) allele nucleotide sequence, said sequences comprising a nucleotide corresponding to the nucleotide at position 2622 of a GPIIb cDNA, and then (ii) analyzing the Bak-allele specificity of said probe that has hybridized to said amplified portion of genomic DNA to determine said Bak allele or alleles.
 17. A method as claimed in claim 13 wherein step (C) comprises the steps of (i) hybridizing said amplified portion of genomic DNA with a pair of oligonucleotide probes to produce a construct, wherein the first probe of said pair is labeled with a first label and the second probe of said pair is labeled with a second label, such that the first label is distinguishable from the second label, and the probes hybridize adjacently to each other at the nucleotide of the amplified portion of the genomic DNA that corresponds to a nucleotide at position 2622 of a GPIIb cDNA; and thereafter (ii) reacting said construct with a ligase in a reaction medium; and then (iii) analyzing said reaction medium to detect the presence of a ligation product comprising said first probe ligated to said second probe. 